The mutant form of Wolinella succinogenes L-asparaginase (WASm) contains two replacements, V23Q and K24T, in the N-terminal flexible loop, which restricts the active center. This form of the enzyme has an order of magnitude lower glutaminase activity compared to the original enzyme (WAS). The 3D structure of WASm has been determined for the apo-form and WASm complexes with L-aspartic and L-glutamic amino acids with a resolution of 1.70, 1.65, and 2.0 Å, respectively. The amino acid residues of the N-terminal flexible loop are only partially localized on electron density maps. In the corresponding complexes, all active centers of the tetrameric enzyme molecules are fully occupied with aspartic and glutamic acids. Their environment has been described, and their location in the original and mutant enzyme coincides. The state of the active centers in the studied molecules has been considered. It has been shown that the active centers in all subunits of the WASm apo-enzyme and WASm/Glu complex are in open conformation, while those in three subunits of the WASm/Asp complex are closed and the conformation is open only in one subunit. The comparison of three-dimensional structures of the original and mutant enzyme complexes suggests that the decrease in glutaminase activity of WASm is caused by the increase in the flexibility of the residues in the N‑terminal loop, which complicates the formation of the catalytically active closed form of the active center after the binding to the less specific substrate (glutamine).

中文翻译：

---

与 L-天冬氨酸和 L-谷氨酸形成复合物的 Wolinella succinogenes L-天冬酰胺酶突变形式的三维结构特征

Wolinella succinogenes L-天冬酰胺酶 (WASm) 的突变形式在 N 端柔性环中包含两个替代物 V23Q 和 K24T，这限制了活性中心。与原始酶 (WAS) 相比，这种形式的酶的谷氨酰胺酶活性低一个数量级。WASm 的 3D 结构已确定为具有 L-天冬氨酸和 L-谷氨酸的 apo 型和 WASm 复合物，分辨率分别为 1.70、1.65 和 2.0 Å。N 端柔性环的氨基酸残基仅部分位于电子密度图上。在相应的复合物中，四聚酶分子的所有活性中心都被天冬氨酸和谷氨酸完全占据。它们的环境已被描述，它们在原始酶和突变酶中的位置重合。已经考虑了所研究分子中活性中心的状态。已经表明，WASm 脱辅基酶和 WASm/Glu 复合物所有亚基的活性中心都处于开放构象，而 WASm/Asp 复合物的三个亚基中的活性中心是封闭的，只有一个亚基的构象是开放的。原始酶复合物和突变酶复合物的三维结构比较表明，WASm 谷氨酰胺酶活性的降低是由于 N 端环中残基的灵活性增加引起的，这使得催化活性闭合的形成复杂化。与特异性较低的底物（谷氨酰胺）结合后形成活性中心。已经表明，WASm 脱辅基酶和 WASm/Glu 复合物所有亚基的活性中心都处于开放构象，而 WASm/Asp 复合物的三个亚基中的活性中心是封闭的，只有一个亚基的构象是开放的。原始酶复合物和突变酶复合物的三维结构比较表明，WASm 谷氨酰胺酶活性的降低是由于 N 端环中残基的灵活性增加引起的，这使得催化活性闭合的形成复杂化。与特异性较低的底物（谷氨酰胺）结合后形成活性中心。已经表明，WASm 脱辅基酶和 WASm/Glu 复合物所有亚基的活性中心都处于开放构象，而 WASm/Asp 复合物的三个亚基中的活性中心是封闭的，只有一个亚基的构象是开放的。原始酶复合物和突变酶复合物的三维结构比较表明，WASm 谷氨酰胺酶活性的降低是由于 N 端环中残基的灵活性增加引起的，这使得催化活性闭合的形成复杂化。与特异性较低的底物（谷氨酰胺）结合后形成活性中心。